The present invention relates generally to the synthesis of aromatic compounds and in particular, those compounds that possess fresh, fruity characteristics and notes. In recent years there have been a strong consumer demand for things that are natural, and this preference is nowhere more prevalent than the choice of natural flavors over synthetic substitutes. In order to successfully fulfill this demand, new methods are required for producing those compounds responsible for these natural aromas and notes.
Grape flavors are always in demand as a flavor component in foodstuffs and beverages and concord grape, in particular, has a unique and pleasant taste and aroma. Methyl anthranilate for example, is known to be major contributor to the aroma of this fruit and is in fact regarded as an impact compound as an essential component for its flavor. It is also often used as an indispensable ingredient in other flavoring systems. This ester is often used industrially in different food flavorings and perfume compositions. Described as a "green apple odor" it has also been identified in several essential oils such as neroli, bergamot and wood strawberry. Although it is widely distributed in nature, it is not readily extracted and economically feasible to isolate due to its low levels when present in a particular source. Ethyl anthranilate is a derivative responsible for peach, berry, grape and orange flavors while butyl anthranilate is an important aromatic in plum flavors and perfumes. Other valuable aromatic ester flavor compounds include methyl salicylate and methyl cinnamate.
Two known methods for the production of natural methyl anthranilate include the bioconversion (demethylation) of dimethyl anthranilate (N-methyl methyl anthranilate) to methyl anthranilate by Trametes and Polyporous fungi and the fermentation thereof by another fungal group Pyconoporous Cinnabarinus. Since the starting material needed in the demethylation reaction of the first process is in limited supply and is actually quite scarce, and the fermentation process only yields very low titres, the cost of natural methyl anthranilate is fairly high.
U.S. Pat. No. 5,200,330 to Page et. al. discloses microbially mediated N-demethylation of dimethyl anthranilate to yield methyl anthranilate using cultures of Trametes versicolor and Polyporous sovalis, two species of fungi. However, not only are the yields of methyl anthranilate low, but a secondary byproduct, N-formyl-methyl anthranilate is also produced. Another problem that exists in attempts to microbially ferment methyl anthranilate through the demethylation of dimethyl anthranilate is that both the starting material and the product are highly toxic to most microorganisms. Moreover, the substrate stereochemistry is problematical because the microbiological demethylations are enzymatic and hence are highly selective.
Gross et. al., App. Microbi. and Biotech. (1990) 34-387-391 reports the synthesis of methyl anthranilate from Pycnoporous cinnabarinus fermentation from a culture medium containing diammonium tartrate, sugars and maltose. Again unfortunately, relatively low yield levels are obtained.
Another possible route is the esterification of anthranilic acid. Chemical esterification involves the reaction of anthranilic acid in methanol with an acid catalyst at high temperatures. This is not considered a natural procedure however, and must be run at high temperatures which is not suitable for many applications. U.S. Pat. No. 3,189,529 to Yamado et. al. discloses a method for the isolation and purification of a high activity lipase from a culture of the yeast Candida cylindraea. Gillies et. al., Biocatalysis in Organic Medium 227 (1986) reported on the production of natural flavor esters such as ethyl butyrate and isoamyl butyrate using this same lipase absorbed onto silica gel.
The present invention provides a means to overcome the problems of low yields while still utilizing a natural process. The present invention more specifically relates to the use of enzymes to esterify natural anthranilic acid, cinnamic acid and salicylic acid to form natural methyl anthranilate, methyl cinnamate and methyl salicylate, respectively. The enzymes found to be of use are able to catalyze the esterification reaction at moderate temperatures which makes it easier to run and is suitable for all applications. And of course, the added benefit remains that the chemicals consumed in the process as well as the methyl anthranilate final product may all be classified as natural.